The binding of streptococci to human platelets is a postulated central mechanism in the pathogenesis of infective endocarditis. Bacterium-platelet binding may be important both for the initial attachment of blood-borne organisms to the valve surface, and for the subsequent formation of macroscopic vegetations. The goal of this project is to characterize further the molecular basis for direct platelet binding by Streptococcus gordonii, and the role of binding in the pathogenesis of endocarditis. We have identified two loci of S. gordonii strain M99 that mediate the direct binding of this organism to human platelets in vitro. The first locus (gspA) encodes a 117 kDa cell wall-associated protein that may function as an adhesin. The second locus appears to be an operon (gspB-secY2A2) encoding a 286 kDa cell wall anchored protein (GspB) that is also a likely platelet binding protein. In addition, this operon contains genes (secA2 and secY2) that are required for the export of GspB. We now seek to further examine the role of these genes and their products in mediating binding to platelets. Our first goal is to characterize more extensively these loci by confirming that expression of gspA or gspB is linked to platelet binding. This work will include complementation studies of gspA and gspB mutants, expression of GspA or GspB in Lactococcus lactis and studying its effects on platelet binding by that organism, and additional mapping of the gspB-secY2A2 operon. We will then purify GspA and GspB, and examine the binding properties of each protein with human platelets in vitro, thereby determining if binding resembles a receptor-ligand interaction. Purified GspA and GspB will also be used to identify their respective platelet binding sites. To assess the role of these adhesins in the pathogenesis of endocarditis, we will compare the virulence of M99 and selected mutants in a rabbit model of endocardial infection. By characterizing streptococcal adhesins for platelets, this research will further define the role of platelet binding in the pathogenesis of endocarditis. In addition, it may identify novel targets for new preventative or therapeutic strategies.